Method to enhance delivery of glutathione and atp levels in cells

ABSTRACT

A therapeutic method is provided comprising treating a mammal subject to hypoxia with an amount of 2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid (RibCys) or a pharmaceutically acceptable salt thereof effective to both maintain, restore or increase both the ATP levels and the glutathione (GSH) levels in said tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/990,933, filed Nov. 17, 2004, which is incorporated herein byreference.

BACKGROUND OF THE INVENTION

The protective mechanisms of mammalian cells against exogeneous andendogenous stressors that generate harmful free radicals employ theantioxidant co-enzyme, glutathione (GSH). GSH is important inmaintaining the structural integrity of cell and organelle membranes andin the synthesis of microtubules and macromolecules. See C. D. Klassenet al. Fundamental and Applied Toxicology, 5, 806 (1985). Stimulation ofGSH synthesis in rat renal epithelial cells and stomach cells has beenfound to protect the cells from the toxic effects of cyclophosphamideand serotonin, respectively. Conversely, inhibition of glutathionesynthesis and glutathione depletion has been found to have the followingeffects: (a) decreased cell viability, (b) increased sensitivity ofcells to the effects or irradiation, (c) increased sensitivity of tumorcells to peroxide cytolysis, (d) decreased synthesis of prostaglandin Eand leukotrienc C and (e) selective destruction of trypanosomes in mice.

Biosynthesis of glutathione (GSH) involves two sequential reactions thatutilize ATP and that are catalyzed by the enzymes γ-glutamylcysteinesynthetase and glutathione synthetase (GSH-synthetase) using the threeprecursor amino acids L-glutamic acid, L-cysteine, and glycine, as shownin FIG. 1.

All substrate-level reactants occur at near enzyme-saturatingconcentrations in vivo with the exception of L-cysteine, whose cellularconcentration is exceedingly low. Therefore, the first reaction in whichL-cysteine is required, i.e., the synthesis of γ-L-glutamyl-L-cysteine,is the rate-limiting step of glutathione biosynthesis. Thus, theavailability of intracellular L-cysteine is a critical factor in theoverall biosynthesis of GSH, are sufficient stores of ATP.

In the synthesis of ATP via the nucleotide salvage pathway, thenucleotide precursors that may be present in the tissue are converted toAMP and further phosphorylated to ATP. Adenosine is directlyphospborylated to AMP, while xanthine and inosine are first ribosylatedby 5-phosphoribosyl-1-pyrophosphate (PRPP) and then converted to AMP.

Ribose is found in the normal diet only in very low amounts, and issynthesized within the body by the pentose phosphate pathway. In the denovo synthetic pathway, ribose is phosphorylated to PRPP, and condensedwith adenine to form the intermediate adenosine monophosphate (AMP). AMPis further phosphorylated via high energy bonds to form adenosinediphosphate (ADP) and ATP.

During energy consumption, ATP loses one high energy bond to form ADP,which can be hydrolyzed to AMP. AMP and its metabolites adenine, inosineand hypoxanthine are freely diffusible from the muscle cell and may notbe available for resynthesis to ATP via the salvage pathway.

The availability of PRPP appears to control the activity of both thesalvage and do novo pathways, as well as the direct conversion ofadenine to ATP. Production of PRPP from glucose via the pentosephosphate pathway appears to be limited by the enzymeglucose-6-phosphate dehydrogenase (G6PDH). Glucose is converted byenzymes such as G6PDH to ribose-5-phosphate and further phosphorylatedto PRPP, which augments the de novo and salvage pathways, as well as theutilization of adenine.

Many conditions produce hypoxia. Such conditions include acute orchronic ischemia when blood flow to the tissue is reduced due tocoronary artery disease or peripheral vascular disease where the arteryis partially blocked by atherosclerotic plaques. In U.S. Pat. No.4,719,201, it is disclosed that when ATP is hydrolyzed to AMP in cardiacmuscle during ischemia, the AMP is further metabolized to adenosine,inosine and hypoxanthine, which are lost from the cell upon reperfusion.In the absence of AMP, rephosphorylation to ADP and ATP cannot takeplace. Since the precursors were washed from the cell, the nucleotidesalvage pathway is not available to replenish ATP levels. It isdisclosed that when ribose is administered via intravenous perfusioninto a heart recovering from ischemia, recovery of ATP levels isenhanced.

Transient hypoxia frequency occurs in individuals undergoing anesthesiaand/or surgical procedures in which blood flow to a tissue istemporarily interrupted. Peripheral vascular disease can be mimicked inintermittent claudication where temporary arterial spasm causes similarsymptoms. Finally, persons undergoing intense physical exercise orencountering high altitudes may become hypoxic. U.S. Pat. No. 6,218,366discloses that tolerance to hypoxia can be increased by theadministration of ribose prior to the hypoxic event.

Hypoxia or ischemia can also deplete GSH. For example, strenuous aerobicexercise can also deplete antioxidants from the skeletal muscles, andsometimes also from the other organs. Exercise increases the body'soxidative burden by calling on the tissues to generate more energy.Making more ATP requires using more oxygen, and this in turn results ingreater production of oxygen free radicals. Studies in humans andanimals indicate GSH is depleted by exercise, and that for the habitualexerciser supplementation with GSH precursors may be effective inmaintaining performance levels. See L. L. Ji, Free Rad. Biol. Med., 18,1079 (1995).

Tissue injury, as from burns, ischemia and reperfusion, surgery, septicshock, or trauma can also deplete tissue GSH. See, e.g., K. Yagi, LipidPeroxides in Biology and Medicine, Academic Press, N.Y. (1982) at pages223-242; A. Blaustein et al., Circulation. 80, 1449 (1989); H. B.Demopoulos, Pathology of Oxygen, A. P. Autor, ed., Academic Press, N.Y.(1982) at pages 127-128; J. Vina et al., Brit. J. Nutr., 421 (1992); C.D. Spies et al., Crit. Care Med. 22, 1738 (1994); B. M. Lomaestro etal., Annals. Pharmacothe., 29, 1263 (1995) and P. M. Kidd, Alt. Mod.Res., 2, 155 (1992).

It has been hypothesized that delivery of L-cysteine to mammalian cellscan elevate GSH levels by supplying this biochemical GSH precursor tothe cell. However, cysteine itself is neurotoxic when administered tomammals, and is rapidly degraded. In previous studies, it was shown thatN-acetyl-L-cysteine, L-2-oxothiazolidine-4-carboxylate, as well as2(R,S)-n-propyl-, 2(R,S)-n-pentyl and2(R,S)-methyl-thiazolidine-4R-carboxylate can protect mice fromheptatotoxic dosages of acetaminophen. See H. T. Nagasawa et al., J. MedChem. 22, 591 (1984) and A. Meister et al., U.S. Pat. No. 4,335,210.L-2-Oxothiazolidinc-4-carboxylate is converted to L-cysteine via theenzyme 5-oxo-L-prolinase. As depicted in FIG. 2, compounds of formula 1,e.g., wherein R=CH₃, function as prodrug forms of L-cysteine (2),liberating this sulfhydryl amino aciuc by nonenzymatic ring opening andhydrolysis. However, the dissociation to yield L-cysteine necessarilyreleases an equimolar amount of the aldehyde (3), RCHO. In prodrugs inwhich R is an aromatic or an alkyl residue, the potential for toxiceffects is present.

U.S. Pat. No. 4,868,114 discloses a method comprising stimulating thebiosynthesis of glutathione in mammalian cells by contacting the cellswith an effective amount of a compound of the formula (1):

wherein R is a (CHOH)_(n)CH₂OH and wherein n is 1-5. The compoundwherein n is 3 is2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid (Ribose-Cysteine, RibCys). Following in vivo administration, RibCysreleases cysteine by non-enzymatic hydrolysis. RibCys has beendemonstrated to be effective to protect against acetaminophen-inducedhepatic and renal toxicity. A. M. Lucus, Toxicol. Pathol., 2, 697(2000). RibCys can also protect the large and small bowel againstradiation injury. See M. P. Caroll et al., Dis. Colon Rectum, 716(1995). These protective effects are believed to be due to thestimulation of GSH biosynthesis, which elevates intracellular GSH.However, a need exists for methods to restore or maintain intracellularGSH stores in mammalian tissues subjected to hypoxic conditions in whichthe ATP stores necessary to drive the biosynthesis of GSH and itsprecursors are depleted.

SUMMARY OF THE INVENTION

The present invention provides a method to treat a mammal threatened by,or afflicted with a hypoxic condition (hypoxia) comprising administeringan effective amount of a compound of formula (Ia):

(RibCys) or a pharmaceutically acceptable salt thereof, effective tocounteract the effects of said hypoxia in the tissue(s) of said mammal.Although depressed glutathione levels have been implicated in a numberof hypoxic conditions, as discussed above, the use of RibCys or itssalts to prevent, counteract or otherwise treat such conditions has notbeen reported. It is believed that simply administering a GSH precursorsuch as cysteine will not be as effective in many instances of hypoxia,when the depletion of ATP stores contributes to inhibition to thebiosynthesis of GSH. As well as functioning as a prodrug for cysteine,administration of effective amounts of RibCys can deliver amounts ofribose to ATP-depleted tissues that stimulate the in vivo synthesis ofATP and that also can stimulate the synthesis of NADPH (nicotinamideadenine dinucleotide phosphate, reduced). This coenzyme supplies theelectrons to glutathione reductase, which in turn recycles oxidized GSHvia GSSG, to free GSH, which resumes its protective role as a cofactorfor antioxidant enzymes in the cell. Optionally, compound (la) can beadministered with an additional amount of free ribose. Preferably,administration will be by oral administration, particularly inprophylactic or pre-loading situations, but parenteral administration,as by injection or infusion, may be necessary in some situations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the metabolic synthesis of glutathione (GSH) fromL-glutanic acid.

FIG. 2 depicts the in vivo dissociation of a compound of formula 1 toyield cysteine and an aldehyde.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term RibCys refers to2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidine-4(R)-carboxylic acid, as well as the 2R or 2S enantiomersof (Ia), and its pharmaceutically acceptable salts. Such salts includealkali metal salts of the carboxylic acid moiety as well as stable acidaddition salts of the NH moiety, including salts of both inorganic andorganic acids, such as citrate, malate, gluconate, glutamate,hydrochloride, hydrosulfate and the like.

As used herein, the term “hypoxia” or “hypoxic condition” is defined tomean a condition in which oxygen in one or more tissues of a mammal islowered below physiologic levels, e.g., to a less than optimal level.Hypoxia also includes conditions in which oxygen levels are lowered intissues due to stress such as aerobic exercise, physical weightpressure, anesthesia, surgery, anemia, acute respiratory distresssyndrome, chronic illness, chronic fatigue syndrome, trauma, burns, skinulcers, cachexia due to cancer and other catabolic states and the like.Hypoxia also includes “ischemia” or “ischemic conditions” in whichtissues are oxygen-deprived due to reduction in blood flow, as due toconstriction in, or blockage of, a blood vessel. Ischemia and/orischemic conditions include those caused by coronary artery disease,cardiomyopathy, including alcoholic cardiomyopathy, angioplasty,stenting, heart surgery such as bypass surgery or heart repair surgery(“open-heart surgery”), organ transplantation, prolonged weight pressureon tissues (pressure ulcers or bedsores), ischemia-reperfusion injurywhich can cause damage to transplanted organs or tissue, and the like.The present invention is effective to treat the GSH and ATP depletiondue to hypoxia and thus to increase a subject's energy level strengthand well-being, even though the underlying cause of the hypoxiccondition, such as viral or bacterial infection, exposure to bacterialor other toxins, low red-cell counts, aging, cancer or continuedexercise, is not affected.

The term “treating” or “treatment” as used herein includes the effectsof RibCys administration to both healthy and patients afflicted withchronic or acute illness and includes inducing protective affects aswell as decreasing at least one symptom of a past or ongoing hypoxiccondition.

Effective doses of RibCys will vary dependent upon the condition, ageand weight of the patient to be treated, the condition to be treated andthe mode of administration. Both cysteine, as released in vivo fromRibCys in animal models, and ribose, as administered directly to humansubjects, have been found to be essentially non-toxic over wide dosageranges. For example, ribose has been reported to increase exercisecapacity in healthy human subjects when taken orally at dosages of 8-10g per day by an adult. See U.S. Pat. No. 6,534,480. RibCys administeredto mice at 8 mmol/kg i.p., increased glutathione levels in numerousorgans, including heart (1.5×) and muscle tissue (2.5×). See, J. C.Roberts, Toxicol. Lett, S, 245 (1991). Likewise, RibCys at 8 mmol/kg hasbeen found to deliver effective protective amounts of cysteine to miceexposed to cyclophosphamide. This dose can deliver about 70-80 g ofribose and about 60-70 g of cysteine to an adult human. See J. C.Roberts, Anticancer Res., 4, 383 (1994). Doses of 2 g/kg RibCys werereported to protect mice against acetaminophen hepatic and renaltoxicity by A M. Lucas et al., Toxicol. Pathol., 20, 697 (2000). Dosesof 1 g/kg RibCys were reported to protect mice againstirradiation-induced bowel injury (see J. K. Rowe et al., Dis. ColonRectum. 36, 681 (1993). J. E. Fuher (U.S. Pat. No. 4,719,201) reportedthat doses of ribose of about 3 g/day for at least 5 days effectivelyrestored and maintained ATP levels in dogs subjected to ischemia (heartattack model), doses that delivered about 550-700 mg/kg of ribose to an30 kg dog.

In clinical practice, these compounds, and the pharmaceuticallyacceptable salts thereof, can be administered in the form of apharmaceutical unit dosage form comprising the active ingredient incombination with a pharmaceutically acceptable carrier, which can be asolid, semi-solid, or liquid diluent. A unit dosage of the compound canalso be administered without a carrier material. Examples ofpharmaceutical preparations include, but are not limited to, tablets,powders, capsules, aqueous solutions, suspensions includingconcentrates, liposomes, and other slow-releasing formulations, as wellas transdermal delivery forms. Typically, the unit dosage form includesabout 0.001-99% of the active substance.

The compounds can be delivered by any suitable means, e.g., topically,orally, parenterally. Preferably, the delivery form is liquid or a solidsuch as a powder that can be stirred into an ingestible liquid. Standardpharmaceutical carriers for topical, oral, or parenteral compositionsmay be used, many of which are described in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa.

For example, for oral administration, suitable pharmaceutical carriersor diluents can include mannitol, lactose, starch, magnesium stearate,talcum, glucose, and magnesium carbonate. Oral compositions can be inthe form of tablets, capsules, powders, solutions, suspensions,sustained release formulations, and the like. A typical tablet orcapsule can contain 40-99% lactose, 1-2% magnesium stearate, and 10-20%cornstarch, along with the active substance (preferably about0.001-20%). An aqueous solution can contain up to the saturation levelof RibCys or its salt, preferably with an amount of ribose added that iseffective to prevent or inhibit premature in vitro dissociation.

For parenteral administration, suitable pharmaceutical carriers caninclude water, saline, dextrose, Hank's solution, Ringer's solution,glycerol, and the like. Parenteral compositions can be in the form ofsuspensions, solutions, emulsions, and the like. Parenteraladministration is usually by injection or infusion which can besubcutaneous, intramuscular, or intravenous.

Example 12(R,S)-D-ribo-1′,2′,3′,4′-Tetrahydroxybutylthiazoidine-4(R)-carboxylicAcid (RibCys)

This compound was synthesized using ribose (Rib) as described by R.Bognar et al., Z. Liebigs Ann. Chem., 738, 68 (1970), the disclosure ofwhich is incorporated by reference herein. The product was collected togive 4.71 g (92.2% yield) of pale yellow material, mp 149°-151° C. dec.[α]_(D) ²⁵—103.1° (c=0.52, H₂O); IR (KBr) v3220 (br, OH, COO⁻), 1610cm⁻¹ (COO).

Example 2 Stimulation of Glutathione Biosynthesis in Isolated RatHepatocytes by L-Cysteine Prodrugs and Inhibition by ButhionineSulfoximine (BSO)

Rat hepatocytes were isolated following the methods of P. O. Seglen,Exper. Cell Res., 74, 450 (1972). After final plating, the hepatocyteswere maintained in culture for 24 hr prior to use. Only primary cultureswere used throughout the studies. The hepatocytes were incubated withcysteine prodrugs NAC and (Ia) for a 4-hr period, and after removal ofmedia by aspiration, the cells were rinsed with cold phosphate-bufferedsaline and deproteinized with 5% sulfosalicylic acid. Total GSH content(GSH+GSSG) was determined by a modification of the DTNB[5,5′-dithiobis(2-nitrobenzoic acid)]glutathione reductase recyclingmethod of F. Tietze, Anal. Biochem., 22, 502 (1969). The GSHconcentration in the sample was quantified by determining the cyclingrate (AOD at 412 nm/min) of the sample. For the inhibition studies withBSO, the cells were pre-exposed to BSO (0.20 mM) before treatment withthe L-cysteine prodrugs.

The results are shown in Table 1, below:

TABLE 1 Increased Glutathione [GSH] Content of Rat Hepatocytes afterIncubation with L-Cysteine Prodrugs Conc. [GSH] ± SE [GSH] Rel. CysteineProdrug (mM) (nmol/10⁶ cells) to Controls Control (none) — 35.4 ± 0.75 1RibCys (Ia) 1.0 61.2 ± 1.52 1.7 N-Acetyl-L-cysteine (NAC) 2.5 45.8 ±1.27 1.3

As can be seen from Table 1, RibCys elevated GSH levels about 1.7-foldrelative to controls in these hepatocytes. N-Acetyl-L-cysteine (NAC),the drug presently used for the clinical treatment of acetaminophenoverdoses, also raised GSH levels by 30% in this system, but required2.5 times the concentration of the thiazolidine prodrugs for comparableelevation. (See L. F. Prescott et al., Brit. Med. J., 2, 1097 (1979); B.J. Lautenburg et al., J. Clin. Invest., 71, 980 (1983) and G. B.Corcoran et al., J. Pharmacol. Exp. Ther., 232, 864 (1985)).

That GSH biosynthesis was stimulated by liberation of its biochemicalprecursor, L-cysteine, from the prodrugs, was indicated by experimentsconducted in the presence of 0.20 mM buthionine sulfoxime (BSO). O. W.Griffith et al., J. Biol. Chem., 24, 7558 (1979), have demonstrated thatBSO is a specific inhibitor of gamma-glutamyl cysteine synthetase, theenzyme responsible for catalyzing the first step in GSH biosynthesis.The data summarized on Table 2, below, demonstrate that GSH levels weredecreased by this inhibitor even in the presence of RibCys, thusproviding evidence that the increased levels of GSH observed were indeeddue to de novo GSH biosynthesis from the L-cysteine provided by thethiazolidine prodrugs.

TABLE 2 Inhibitory Effect of Buthionine Sulfoxime (BSO) on GSH ElevationElicited by L-Cysteine Prodrugs in Rat Hepatocytes Prodrug BSO [GSH] ±SE [GSH] Rel. (1.0 mM) (0.2 mM) (nmol/10⁶ cells) to Controls. None(Control) − 35.4 ± 0.78 1.0 None + 18.4 ± 2.08 0.5 RibCys (1a) + 16.2 ±3.60 0.5 N-Acetyl-L-cysteine + 25.5 ± 1.59 0.7

Example 3 RibCys Elevates GSH in Heart and Muscle Tissue

As reported by J. C. Roberts et al., Toxicol. Lett., 5, 245 (1991),RibCys successfully elevated glutathione (GSH) levels in numerous organsof tumor-bearing CDF1 mice. GSH content was assayed 1, 2, 4, 8 and 16 hafter RibCys administration (8 mmol/kg, i.p.); various organs achievedmaximal GSH content at different time points. GSH in the liver waselevated 1.5-fold compared to untreated controls at the 16-h time point.Kidney GSH also was maximal at 16 h and achieved 1.6-times controlvalues. GSH in muscle achieved 2.5 times the levels in control animals,while the bladder was elevated 2.1-fold, and the heart 1.8-fold. Othertissues tested (spleen, pancreas, lung) showed a 1.1- to 1.2-foldincrease in GSH content. GSH in implanted L1210 tumors was also elevatedonly 1.2-fold.

Example 4 Recovery of the Working Canine Heart Following GlobalMyocardial Ischemia

As reported in Examples 1-2 of J. E. Foker (U.S. Pat. No. 4,605,644),dilute solutions of ribose in normal (0.9%) saline were found effectiveto decrease the ATP recovery time following myocardial ischemia in thecanine model. For example, infusion of a normal saline solution which is80 mM in ribose at a rate of about 1 ml/min for about 24.0 hoursafforded an eight-fold decrease in the ATP recovery time. During thistreatment period, about 17.0 g of ribose were introduced into thecirculatory system; a total dose of about 550-700 mg ribose/kg of bodyweight. The appropriate dose for the optimal recovery of ATP levels andcardiac function in a given human subject can be readily established viaempirical studies including known assays for ATP levels, cardiacfunction and the like.

Although the studies of the examples of U.S. Pat. No. 4,605,644 weredirected at enhancing the energetic recovery following ischemia of theheart with solutions containing free ribose, the present methodemploying the cysteine/ribose pro-drug RibCys is also expected to beapplicable to any tissue or organ that has suffered hypoxia, such as anischemic insult where antioxidant augmentation and ATP recovery would behelpful. These situations include but are not limited to: myocardialinfarction, stroke, organ transplant with organ preservation, neonatalsupport, multi-organ system failures, shock and trauma resulting incompromised circulation, and the like. Often, even uncomplicated generalanesthesia can result in some degree of hypoxia and the accompanyinginvasive medical procedure can lead to the build-up of free radicals inthe traumatized tissue. Likewise, aerobic exercise in convalescent orhealthy individuals can lead to ATP depletion and the build-up of freeradicals from environmental oxidants. Therefore, the present inventionprovides a method whereby hypoxic tissue can be treated so as to quicklyregain and maintain normal ATP levels, both to improve tissue survivaland to hasten general bodily recovery.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

1-22. (canceled)
 23. A method of maintaining normal ATP and glutathionelevels in a mammal in need thereof comprising administering an effectiveamount of2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid, or a pharmaceutically acceptable salt thereof, to said mammal,wherein said2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid maintains normal ATP and glutathione levels in the mammal in needthereof.
 24. The method of claim 23, wherein said2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid is administered orally, parenterally, intravenously, orintraperitoneally.
 25. The method of claim 23, wherein said2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid is administered orally.
 26. The method of claim 23, wherein said2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid is administered intravenously.
 27. The method of claim 23, whereinsaid mammal has a tissue injury.
 28. The method of claim 23, whereinsaid2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid is administered in a unit dosage form comprising 20% (w/w) ofRibCys.
 29. The method of claim 23, wherein said2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid is administered in a liquid.
 30. The method of claim 29, whereinsaid liquid further comprises an effective amount of ribose to inhibitpremature dissociation of the2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid.
 31. The method of claim 30, wherein said liquid comprises aneffective amount of ribose to inhibit premature in vitro dissociation ofthe2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid.
 32. The method of claim 23, wherein said method consists ofadministering an effective amount of2(R,S)-D-ribo-(1′,2′,3′,4′-tetrahydroxybutyl)thiazolidone-4(R)-carboxylicacid or a pharmaceutically acceptable salt thereof to said mammal.